Electron microscopic studies of adenovirus preparations have revealed the presence of small "virus-like" particles approximately 22 m,4 in diameter in a number of human and simian adenovirus types.1-5 Some workers have interpreted these particles as being adenovirus subunits,4-6 while others1-3 have indicated that they represent a contaminating virus. Our early studies1 suggested that the 22-mA particles were viruses which replicated poorly, if at all, in human cells in the absence of adenovirus replication. Atchison et al.2 also presented evidence that the small particles were infectious, that they were defective, and that they were immunologically distinct from any known adenovirus antigen. They tentatively referred to this virus as adeno-associated virus (AAV) and we shall continue to use this name.The purpose of the present report is to describe some of the physical, biological, and immunological characteristics of the small particles found in various adenovirus preparations with further evidence that they are defective viruses and not adenovirus subunits.Materials and Methods.-Viruses: Two stock pools of adenovirus type 7 (Ad. 7), strain LLE46+, were prepared after five passages in African green monkey kidney (AGMK) cell culture7-9 and one human embryonic kidney (HEK) cell culture passage. These pools had adenovirus infectivity titers in HEK cells of 104'. to 105-5 TCID,5 per 0.1 ml and, when observed under the electron microscope (EM), contained 102 to 108 times as many small virus particles as typical adenovirus particles. These pools also had the capacity to induce SV40 T antigen in HEK and AGMK cells.8' 9 This substrain shall be called E46 + (+1), the symbol (+1) indicating presence of AAV-1 particles.E46-, a substrain of LLE46 + obtained by limiting dilution passage in HEK cells,8 was grown in HEK cells. This substrain had lost the capacity to induce SV40 T antigen in tissue culture, and was free of AAV. It will be referred to as E46-(0), the symbol (0) indicating absence of small particles. The E46-(0) pool had an infectivity titer of 108 TCID5o per 0.1 ml in HEK cells.The presence or absence of SV40 T antigen was checked by immunofluorescence and AAV was checked by complement fixation (CF), immunofluorescence, and EM.A pool of adenovirus type 12, strain 97838, was found contaminated with a serologically distinct AAV.10 This AAV is referred to as AAV-2. AAV-2 was readily transferred to E46-(0) which then became E46 -(+2).A third AAV type (AAV-3) was recovered from a passage of E46 + in AGI\IK cells in the presence of AAV-1 antiserum.A pool of infectious canine hepatitis virus (ICH) made from a field strain" was examined and found free of small particles. This ICH (0) pool had an infectivity titer in Madin-Darby continuous canine kidney cells (MDCK),'2 of 106.5 TCID5, per 0.1 ml.Assays of adenovirus infectivity were made in HEK cells using 2-3 tubes per dilution. AAV infectivity was measured by titration in HEK cells in the presence of 10' TCID,5o of E46-(0), allowing the cultures to progress to 4+ cyto...
